D-Methionine and 2-hydroxy-4-methylthiobutanoic acid i alter beta-casein, proteins and metabolites linked in milk protein synthesis in bovine mammary epithelial cells

Abstract

This study aims to determine the effects of D-methionine (D-Met) isomer and the methionine precursor 2-hydroxy-4-methylthiobutanoic acid i (HMBi) supplementation on milk protein synthesis on immortalized bovine mammary epithelial cell (MAC-T). MAC-T cells were seeded using 10-cm dishes and cultured in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) basic medium. The basic medium of DMEM/F12 was replaced with the lactogenic DMEM/F12 differentiation medium when 90% of MAC-T cells reached confluency. The best dosage at 0.6 mM of D-Met and HMBi and incubation time at 72 h were used uniformly for all treatments. Each treatment was replicated six times wherein treatments were randomly assigned in a 6-well plate. Cell, medium, and total protein were determined using a bicinchoninic acid protein assay kit. Genes, proteomics and metabolomics analyses were also done to determine the mechanism of the milk protein synthesis pathway. Data were analyzed by two-way analysis of variance (ANOVA) with supplement type and plate as fixed effects. The least significant difference test was used to evaluate the differences among treatments. The HMBi treatment group had the highest beta-casein and S6 kinase beta-1 (S6K1) mRNA gene expression levels. HMBi and D-Met treatments have higher gene expressions compared to the control group. In terms of medium protein content, HMBi had a higher medium protein quantity than the control although not significantly different from the D-Met group. HMBi supplementation stimulated the production of eukaryotic translation initiation factor 3 subunit protein essential for protein translation initiation resulting in higher medium protein synthesis in the HMBi group than in the control group. The protein pathway analysis results showed that the D-Met group stimulated fructose–galactose metabolism, glycolysis pathway, phosphoinositide 3 kinase, and pyruvate metabolism. The HMBi group stimulated the pentose phosphate and glycolysis pathways. Metabolite analysis revealed that the D-Met treatment group increased seven metabolites and decreased uridine monophosphate (UMP) production. HMBi supplementation increased the production of three metabolites and decreased UMP and N-acetyl-L-glutamate production. Taken together, D-Met and HMBi supplementation are effective in stimulating milk protein synthesis in MAC-T cells by genes, proteins, and metabolites stimulation linked to milk protein synthesis.

Dietary L-Methionine modulates the gut microbiota and improves the expression of tight junctions in an in vitro model of the chicken gastrointestinal tract

“…Methionine is an essential amino acid that has been widely utilized in the animal realm as a supplement for better growth performance [ 19 ]. It is necessary for life and must be ingested since animals cannot synthesize methionine by themselves in order to produce the necessary quantity of proteins.…”

“…Generally, there are two isomers (L- and D-form) of amino acids, and in order to be utilized in the animal’s body, D-isomer amino acids must be converted to the L-form mainly in the liver and kidney [ 16 ]. In the poultry industry, the L-form of Met (L-Met) and the combination of D-form and L-form of Met (DL-Met) have been mostly employed as a Met source in chicken feeds, and the ratio of the L-form and D-from has determined its bioavailability and functionality to regulate physiology and metabolism in the animal’s body [ 17 – 19 ]. D- and L-form methionine are utilized indiscriminately as animal feed in the domain.…”

Multi-Omics Approaches to Improve Meat Quality and Taste Characteristics

“…Proteomics uses various techniques such as electrophoresis, protein sequencing, and image statistics to visually describe the expression and function of a proteome. Proteomics tools currently used in scientific research usually include examination of protein expressions, modifications, or interactions on a large scale ( Jeon et al, 2022 ; Kim et al, 2023c ; Seo and Oh, 2023 ). In short, proteomics is a study that identifies and determines types and abundance of proteins, explains protein-protein interaction, and explores localization of proteins in cells and post-translational modification ( Go et al, 2022 ; Jorrín-Novo et al, 2015 ; Jung et al, 2022 ; Kim et al, 2022b ).…”